tbuckley/cabot-search huggingface dataset:
Abstract P1081: Palmitoylation-dependent Regulation Of Rab3gap1, Cardiomyocyte Exocytosis, And Atrial Natriuretic Peptide Secretion
Authors: Kobina Essandoh, Arasakumar Subramani, Matthew Brody
Journal: Circulation Research (2022)

Palmitoylation is the reversible post-translational modification of proteins with fatty acids catalyzed by zDHHC S-acyltransferases that functions as a potent regulatory mechanism controlling protein trafficking and signal transduction. To investigate functions of palmitoylation in cardiac homeostasis and pathophysiology, we generated transgenic mice with cardiomyocyte-specific overexpression of the Golgi enzyme, zDHHC9, which was previously reported as the Ras S-acyltransferase and is associated with X-linked intellectual disability. Although we did not observe altered Ras palmitoylation, zDHHC9 transgenic mice develop age-dependent cardiac hypertrophy that progresses to functional decompensation and cardiomyopathy. We found palmitoylation of Rab3 GTPase activating protein 1 (Rab3gap1) was significantly elevated in zDHHC9 overexpressing hearts prior to disease onset concomitant with enhanced GTP-loading of Rab3a, suggesting zDHHC9-mediated palmitoylation impairs Rab3gap1-dependent nucleotide cycling on Rab3a. Consistent with this, zDHHC9 overexpression resulted in enhanced Rab3gap1 retention at the Golgi and expansion of Rab3a-positive vesicles at the cardiomyocyte periphery. Co-immunoprecipitation analysis in HEK293AD cells confirmed that zDHHC9 elevation diminishes interaction between active Rab3a and Rab3gap1. To interrogate roles of zDHHC9 in cardiomyocyte exocytosis, we assessed secretion of atrial natriuretic peptide (ANP) and surprisingly found that while zDHHC9 overexpression increased intracellular retention and reduced release of ANP, knockdown of zDHHC9 promoted phenylephrine-induced ANP secretion. Taken together, these data suggest zDHHC9-mediated palmitoylation of Rab3gap1 represses ANP secretion by disrupting GTP:GDP cycling on Rab3a necessary for release of ANP from secretory granules. These studies reveal unexpected functions for zDHHC9-mediated palmitoylation in modulation of secretory pathway flux and regulated hormone secretion via downstream impacts on Rab3a-mediated vesicle fusion and exocytosis and suggest novel roles for zDHHC9, Rab3gap1, and Rab3a in natriuretic peptide secretion.
Abstract GS111: Inhibition Of Cardiac Glucose Transporter 1 Suppresses Early Glucose Dependency And Klf5 Activation And Treats Cardiomyopathy In Diabetes
Authors: Sobuj Mia, Ioannis D Kyriazis, Rafailia Sidiropoulou, Daniel Hill, Athanasios Mantalaris, Konstantinos Drosatos
Journal: Circulation Research (2022)

Healthy hearts use more fatty acids (FA) than glucose for ATP synthesis but diabetic cardiomyopathy (DbCM) occurs with higher FA dependency. It remains controversial whether glucotoxicity or lipotoxicity or both account for DbCM. We recently discovered that insulin signaling inhibition and eventual FOXO1 activation stimulate cardiac KLF5 expression, which drives lipotoxicity and causes cardiac dysfunction. In the present study, we investigated the relative contribution of glucose in the activation of cardiac KLF5 and DbCM. We induced Type-1 diabetes (T1D) in C57BL/6 mice via intraperitoneal injections of streptozotocin (STZ). In contrast to late-stage diabetes (12 weeks post-STZ), cardiac KLF5 mRNA and protein levels were not increased in the early T1D stage (4 weeks post-STZ) although mice have mild cardiac dysfunction already. Seahorse analysis in adult cardiomyocytes isolated from mice with early T1D showed higher glucose and lower FA dependency compared to non-diabetic mice and mice in late T1D. To confirm whether hyperglycemia causes cardiac dysfunction, we treated diabetic mice with Dapagliflozin (DAPA, SGLT2 inhibitor) or STF-31, a GLUT1 inhibitor. These treatments restored normal dependency on fatty acids and prevented cardiac dysfunction. GC-MS analysis showed that the reversal of fuel dependency from glucose to fatty acids in late T1D is accompanied by increased glucose content opposite to the early T1D. Accordingly cardiac KLF5 is increased in late T1D, accompanied by severe cardiac dysfunction. The expression changes of KLF5 are mirrored by transcriptional activity of FOXO1 -shown by expression of FOXO1 targets- in early and late T1D. The changes in transcriptional activity are accompanied by differential FOXO1 acetylation, which is controlled by Sirtuin-1 and modulates its DNA affinity. Analysis of mouse cardiac tissue in early T1D and a human cardiomyocyte cell line (AC16) that was treated with high glucose showed higher Sirtuin-1 expression and stronger protein-protein interaction with FOXO1. To this end, mice that were subjected to treatment with either DAPA or STF31 for 12 Wks had improved cardiac function, lower cardiac KLF5 expression and decreased expression of cardiac KLF5 gene targets. Interestingly, GLUT1 mRNA levels were increased in late T1D compared to early T1D. Cardiomyocyte-specific KLF5 overexpression or adenovirus-mediated KLF5 overexpression in AC16 cells stimulated GLUT1 expression. Collectively, in early T1D, hearts rely more on glucose utilization in mitochondria. In late T1D, SIRT1-FOXO1-KLF5 axis causes lipotoxicity and subsequent induction of GLUT1 expression that contributes to glucotoxicity. Inhibition of GLUT1-dependent glucose uptake alleviates diabetic cardiomyopathy via inhibition of both early glucose dependency and late KLF5 activation.
Snoring or bubbling?
Authors: E Lens
Journal: European Respiratory Journal (1989)

conducted an interesting study about snoring in a dog-model [1].The authors speculated about the possibility of "Bubbling" which "may contribute to the sound of snoring".At the Thirteenth "International Conference On Lung Sounds" held, in Chicago, September 1988 we showed our results of spectral analysis of 1565 samples of snores recorded at night with the microphone attached to the stemal notch of patients with asthma, chronic obstructive pulmonary disease, obesity and sleep apnea syndrome, myxoedema, tonsillitis and apneic and non apneic snoring [2).We systematized a classification of the different acoustic shapes of snoring.The figure shows the repetitive character of all snores which are different from the random merging of various crackles in vibration of secretions or bubbling.Thus, spectral analysis may provide an accurate help to differentiate snoring from bubbling.
Abstract P2028: A Role For Pannexin 1 In Heart Failure Induced By Acute And Chronic Isoproterenol Administration
Authors: Caitlin M Pavelec, Matthew J Wolf, Scott Yeudall, Clint Upchurch, Norbert Leitinger
Journal: Circulation Research (2022)

Pannexin 1 (Panx1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation and blood pressure regulation. Panx1 channel function is inhibited by spironolactone, a frontline therapy for heart failure patients. Despite this, the function of Panx1 in cardiomyocytes and a possible role in heart failure has not yet been studied. To investigate if Panx1 is involved in the development of cardiac dysfunction and fibrosis, we use an acute (14 day) and a chronic (28 day) model of adrenergic stimulation to induce heart failure in two novel mouse models: 1) We have generated mice with constitutive deletion of Panx1 in cardiomyocytes by crossing mice expressing loxP-flanked alleles of Panx1 to mice expressing Cre recombinase under the control of the cardiomyocyte specific MyHC6 promoter (Panx1 MyHC6-Cre ). 2) Using mice expressing the tamoxifen-inducible MerCreMer system under the control of the Tnnt2 promoter we generated mice with tamoxifen-inducible deletion of Panx1 in cardiomyocytes (Panx1 Tnnt2-MerCreMer ). Constitutive deletion of Panx1 in cardiomyocytes had no effect on heart function at baseline as measured by echocardiography. However, after acute isoproterenol treatment (15 mg/kg/day, i.p.), Panx1 MyHC6-Cre mice were protected from systolic and diastolic dysfunction and cardiac hypertrophy. Furthermore, after chronic administration of isoproterenol (15 mg/kg/day, osmotic pump) Panx1 MyHC6-Cre mice were still protected from cardiac hypertrophy compared to Panx1 fl/fl littermates. In vitro , treatment of H9c2 cardiomyocytes with isoproterenol led to Panx1-dependent release of ATP in a dose-dependent manner. This was demonstrated with both pharmacological blockade by spironolactone and siRNA-mediated knock-down of Panx1. Taken together, these data demonstrates that Panx1 deficiency in cardiomyocytes protects against isoproterenol-induced cardiac dysfunction and that ATP is released through Panx1 in response to isoproterenol. Further elucidating the role of Panx1 in cardiomyocytes in regulating cardiac function and fibrosis will identify Panx1 as a novel therapeutic target to prevent heart failure.
Abstract P3136: <i>Notch3</i> Deficiency Impairs Vascular Smooth Muscle Cell Contractility And Glymphatic Function In The Brain
Authors: Milagros Romay, Feiyang Ma, Gloria E Hernandez, Jocelynda Salvador, Snezana Mirkov, Ayush Batra, David Sullivan, Russell Knutsen, Elise Kronquist, Elisa Ferrante, Matteo Pellegrini, William A Muller, Manfred Boehm, Beth Kozel, Luisa I Iruela Arispe
Journal: Circulation Research (2022)

While most cases of vascular dementia represent complex interactions between host genetics and environmental factors, mendelian forms of vascular dementia also exist. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), is a mendelian disease characterized by progressive vascular deterioration, cognitive deficits, and strokes. Mutations in the NOTCH3 receptor underlies the pathologies in CADASIL. NOTCH3 is primarily expressed in vascular smooth muscle cells (vSMCs) and its’ expression is critical for differentiation and functional integrity of arterial vSMCs, albeit through unclear mechanism(s). To elucidate the contribution of NOTCH3 in the maintenance of cerebral vascular architecture and function, we performed micro-computed tomography (micro-CT) on the brains of aged Notch3 -deficient animals. Micro-CT assessment of the cerebral vasculature architecture showed significant abnormalities including severe vessel dilation and tortuosity (dolicoectasia) of the middle cerebral artery and its branches in the Notch3 -/- compared to aged-match controls. To identify the molecular pathway from NOTCH3 dysregulation to the observed cerebral vascular dysfunction, we performed single-cell RNASeq on cerebral arteries isolated from young (4w) and old (104w) Notch3 -/- animals. Evaluation of the vSMC-specific transcriptomes indicated significant loss of proteins associated with muscle contraction and increased extracellular matrix production in animals that lack NOTCH3. Using a combination of immunofluorescence microscopy and in vitro functional assays, we confirmed that continued expression of Notch3 is a critical requirement for maintenance of vSMC contractile function. Impaired contractility also affected flow of cerebrospinal fluid in the parenchyma of Notch3 -/- . MRI and behavioral assessments were performed in the Notch3 -/- animals to elucidate the relationship between impaired vascular contractility to cognitive function. Taken together these findings link the molecular dysfunction of NOTCH3 through its regulation of vascular contractility and cerebral vessel architecture to altered neurological function and clarify the molecular pathways to cellular pathology of Notch3 driven dementias.
Abstract P1030: LIN28a-induced Metabolic Reprogramming Regulates New Myocyte Formation In The Heart Via Lncrna-H19
Authors: Vagner O Rigaud, Robert Hoy, Justin Kurian, Clare Zarka, Michael Behanan, Isabella Brosious, Jennifer Pennise, Tej K Patel, Lindsay Kraus, Sadia Mohsin, Steven R Houser, Mohsin Khan
Journal: Circulation Research (2022)

Developmental cardiac tissue holds remarkable capacity to regenerate after injury and consists of regenerative mononuclear and diploid cardiomyocytes (MNDCMs). Upon maturation, MNDCMs become binucleated or polyploid and exit the cell cycle. Interestingly, CM metabolism undergoes a profound shift that coincides with cessation of regeneration in the postnatal heart. However, whether reprogramming metabolism promotes persistence of regenerative MNDCMs enhancing cardiac function and repair after injury is unknown. Here, we identify a novel role for RNA-binding protein LIN28a, a master regulator of cellular metabolism, in cardiac repair following injury. LIN28a was found as primarily active during cardiac development and rapidly decreases after birth. LIN28a reintroduction at P1, P3, P5, and P7 decreased maturation-associated polyploidization, nucleation, and cell size, enhancing CM cell cycle activity in LIN28a transgenic pups compared to WT littermates. Moreover, LIN28a overexpression extended CM cell cycle activity beyond P7 concurrent with increased cardiac function 30 days after apical resection. In the adult heart, LIN28a overexpression attenuated CM apoptosis, enhanced cell cycle activity, cardiac function, and survival in mice 12 weeks after myocardial infarction compared to WT littermate controls. Alternatively, LIN28a small molecule inhibitor attenuated pro-reparative effects of LIN28a on the heart. Mechanistically, Neonatal rat ventricular myocytes (NRVMs) overexpressing LIN28a showed increased glycolysis, ATP production and levels of metabolic enzymes compared to control. LIN28a immunoprecipitation followed by RNA sequencing (RIPseq) in CMs isolated from LIN28a injured hearts identified lncRNA-H19 as its most significantly altered target. Ablation of lncRNA-H19 blunted LIN28a-induced enhancement on CM metabolism and cell cycle activity. Collectively, LIN28a reprograms CM metabolism and promotes persistence of MNDCMs in the injured heart enhancing pro-reparative processes thereby linking CM metabolism to regulation of ploidy/nucleation and repair in the heart.
Abstract P3072: Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (iPSC-CMs) Respond To Inflammatory Stimuli Through Innate Immune Responses
Authors: Daniel F Selgrade, Anthony Gacita, Dominic Fullenkamp, Megan Puckelwartz, Alexis Demonbreun, Marcelo A Nobrega, Alfred L George, Elizabeth M McNally
Journal: Circulation Research (2022)

Introduction: Cells respond to inflammatory stimuli in settings of sterile and pathologic cardiac inflammation. Nuclear factor Kappa B (NFKB) is a transcriptional mediator of innate immune responses. Lipopolysaccharide (LPS) promotes NFKB activation through toll-like receptors (TLR) to initiate cell autonomous processes that resist infection. High-mobility box group 1 (HMGB1) is a chromatin associated protein released upon nuclear rupture that similarly promotes cytokine release in response to injury. Few studies have interrogated multiple inflammatory stimuli and their impacts on iPSC-CM function. We examined innate immune activation using distinct TLR agonists and assessed functional responses in both 2D iPSC-CM monoculture and 3D collagen-based engineered heart tissues. Methods and Results: We measured electric field potential duration (Nanion CardioExcyte 96) of iPSC-CM monolayers after exposure to LPS and HMGB1. We observed a four fold increase in downstroke velocity after exposure to HMGB1 (512.7±126.9 V/s; p=0.0002 n=12) and a 30% decrease in downstroke velocity after LPS treatment (-188.9±61.4 V/s; p=0.004 n=12). This response was associated with nuclear localization of phosphorylated NFKB. Collagen-based engineered heart tissues demonstrated contractile defects in response to 48h of exposure to TLR agonists. A 50% reduction in fractional shortening was observed following HMGB1 treatment (-0.0060±0.0009; p&lt;0.0001 n=6) and an 80% reduction was associated with LPS exposure (-0.0088±0.0006; p&lt;0.0001 n=6). To identify gene expression regulatory regions associated with this response, we examined transcriptional activity of three candidate enhancer regions implicated in cytokine production in iPSC-CMs. These regions demonstrated a 0.5- to 20-fold increase in enhancer activity in iPSC-CMs. One enhancer demonstrated a 22% increase in activity following 48h of LPS, consistent with a direct role for NFKB in innate immune activation in human iPSC-CMs; this enhancer regulates a gene with increased expression in dilated cardiomyopathy. Conclusion: These data indicate that cardiomyocytes respond directly to inflammatory stimuli and demonstrate the utility of 2D and 3D human tissue culture models for cardiac inflammation.
Abstract P1137: Combination Of Zebularine Treated Rat Bone Marrow Mesenchymal Stem Cells With 3d Collagen Scaffold Preserves Ventricular Remodeling In Rat Myocardial Infarction Model
Authors: Rida-e-Maria Qazi, Irfan Khan, Kanwal Haneef, Tuba S Malick, Nadia Naeem, Waqas Ahmad, Asmat Salim, Sadia Mohsin
Journal: Circulation Research (2022)

Cardiovascular diseases (CVDs) are the leading cause of death globally due to immensely high morbidity and mortality ratio. The incidence of CVD, particularly myocardial infarction (MI), is expected to increase rapidly in the coming decades. MI causes the death of myocytes, which ultimately leads to heart failure. Advancements in medical and surgical treatments can only limit the disease progress but cannot improve the function of infarcted myocardium. Therefore, a promising treatment strategy for ischemic heart diseases is highly needed. Stem cell therapy holds great potential to regenerate the injured myocardium due to the self-renewal and differentiation potential of these cells. Among various types, mesenchymal stem cells (MSCs) are potential candidates for regenerating the damaged cardiac tissue due to their multi-lineage differentiation potential and ease of isolation. However, certain issues such as poor survival and engraftment can prevent them from fully restoring normal cardiac structure and function. The use of biomaterials provides suitable microenvironment to the cells to perform various biological functions, and can solve the poor cellular engraftment problem associated with cell transplantation alone. In this study, we used natural three dimensional (3D) collagen scaffold along with demethylating agent, zebularine, for in vivo transplantation in rat MI model. Treatment with zebularine is shown to enhance cardiac differentiation of MSCs. MSCs were isolated from rat bone marrow, characterized, seeded in collagen scaffold and treated with zebularine. Cytotoxicity analysis was performed to confirm the non-cytotoxic effect of the treatments, and gene and protein expression levels were analyzed using cardiac specific primers and antibodies, respectively. In vitro analysis revealed that treatment in the 3D environment significantly enhanced cardiac differentiation of MSCs both at gene and protein levels. For in vivo analysis, rat MI model was developed and zebularine treated MSC-seeded scaffold was transplanted and compared with the MI group, along with other groups in which only collagen scaffold and untreated MSC-seeded scaffold were transplanted. Echocardiographic analysis revealed enhanced functional improvement in zebularine treated MSC-seeded group as compared to the other groups. Histological analysis revealed tissue preservation, cardiac regeneration beneath the scaffold, reduction in the fibrotic scar and improvement in the ventricular wall thickness in the treated MSC group. This strategy is expected to overcome the problems associated with cell transplantation alone and to provide an effective therapeutic strategy for improved cardiac function.
Abstract P1090: Polycystin-1 Regulates Calcium Handling And Contractility In Human IPSC-derived Ventricular Cardiomyocytes
Authors: William Perez, Troy Hendrickson, Francisco J Altamirano
Journal: Circulation Research (2022)

Mutations in polycystin-1 (PC1) cause 85% of autosomal dominant polycystic kidney disease cases. This disease is characterized by progressive renal disease and multiple cardiovascular alterations. Evidence suggests that cardiovascular disease precedes the onset of renal failure. We recently demonstrated that PC1 ablation shortens cardiomyocyte action potential, decreases Ca 2+ transient amplitude, and reduces contractility in adult cardiomyocytes isolated from cardiomyocyte-specific PC1 knock-out mice compared to control mice. Alterations in Ca 2+ cycling in PC1-deficient cardiomyocytes were demonstrated to be due to action potential duration shortening and impaired sarcoendoplasmic reticulum ATPase activity. However, mouse and human cardiomyocytes have considerable differences in their action potential shapes and the ion channels involved. It remains unknown whether PC1 regulates these events in human cardiomyocytes. Here, we sought to investigate whether PC1 regulates excitation-contraction coupling in human ventricular cardiomyocytes. We differentiated commercially available human-induced pluripotent stem cell lines (hiPSC) into ventricular cardiomyocytes (hiPSC-CM) using published protocols (measurements were performed 60-90 days after differentiation). We developed a protocol to simultaneously measure action potentials (FluoVolt), intracellular Ca 2+ (Calbryte-590) and contractility using the IonOptix algorithm to detect changes in contrast as contractility measurements. PC1 ablation using siRNA decreased action potential duration and impaired Ca 2+ transients and contractility in hiPSC-CMs. Because hiPSC-CMs have several limitations due to their immature state, we employed a well-validated method to improve their maturation that utilizes a Matrigel mattress and hormone supplementation (dexamethasone; Dexa / thyroid hormone; T3; PMID: 28974554; developed by Dr. Bjorn Knollmann’s lab). Using the Matrigel mattress/Dexa/T3 technique, we increased hiPSC-CM maturation measured as follows: cardiomyocytes were oriented and contracted in the longitudinal axis, ryanodine receptor type 2 and alpha-actinin localized with a striated pattern, exhibited robust inward K + rectifier currents (I K1 ) and fired action potentials that resemble human adult cardiomyocytes. In this model, PC1 knockdown also decreased action potential duration and impaired Ca 2+ transients and decreased contractility measured as fractional shortening. Altogether, this evidence shows for the first time that PC1 regulates human cardiomyocyte physiology and suggests that cardiac alterations in patients with autosomal dominant polycystic kidney disease may emerge as a result of impaired cardiomyocyte contractile function.